Incorporation of dansylated phospholipids and dehydroergosterol into membranes using a phospholipid exchange protein

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Abstract
A nonspecific phospholipid exchange protein (PLEP) preparation [bovine liver] was used to transfer dansyl[3H]phosphatidylethanolamine (DNS-PE), dansyl[3H]phosphatidylserine (DNS-PS), and dehydroergosterol (DHE) from sonicated lipid veislces to [Electrophorus electricus] electroplax plasma membrane fragments enriched in Na+,K+-ATPase with retention of 80-90% of Na+,K+-ATPase activity. The transfer of individual fluorescent lipid molecules was distinguished from a nonspecific association of lipid vesicles and membranes by including [14C]triolein, a lipid that is not transferred by PLEP, in the vesicles. Dansyl 3H-labeled phospholipids (DNS-[3H]PL) or DHE was considered incorporated into the Na+,K+-ATPase membranes when fluorophores pelleted with the Na+,K+-ATPase preparation without the nonexchangeable [14C]triolein. The locations of incorporated DHE and DNS-PL were also described by iodide quenching experiments. DHE was not accessible to iodide for quenching, while 75% of the DNS-PL incorporated into Na+,K+-ATPase membrane fragments were accessible to iodide. After a technique was developed using PLEP to incorporate fluorescent lipids into membranes with the Na+,K+-ATPase preparation, DNS-PE, DNS-PS, and DHE were then analogously incorporated into electroplax plasma membranes enriched in acetylcholinesterase (AChE) and into [human] erythrocyte ghosts in order to evaluate the fluorophores as membrane probes. In the subseuqent evaluation, the fluorescent properties of membrane-incorporated DNS-PE, DNS-PS, and DHE were systematically compared to the fluorescent properties of the molecules in lipid vesicles. The fluorescence polarizations of both DNS-PL were increased by the presence of protein in a bilayer. The fluorescence polarization of DNS-PS was greater than the polarization of DNS-PE in both membranes and vesicles. In contrast, the polarization (and the lifetime) of DHE was the same whether the fluorescent sterol was in a membrane preparation or in vesicles. Fluorescence polarization and intensity of all 3 fluorophores were measured in the bilayer preparations as a function of temperature. The intensities of all 3 probes and the polarization of DNS-PE in both membranes and vesicles decreased biphasically with a change in slope occurring at 26.0.degree.-27.5.degree. C. DNS-PS in lipid vesicles was depolarized biphasically with increasing temperature, but when incorporated into membranes, DNS-PS was depolarized linearly without a change in slope. The polarization of DHE in either membranous or vesicle bilayers did not change with temperature. When the Na+,K+-ATPase membrane preparation was used as a model system, the question was asked whether or not the fluorescent parameters of the lipid probes (i.e., intensity, polarization, and lifetime) could detect conformational changes in an intrinsic membrane enzyme. Results were negative despite localization of the fluorophores near the protein.